Citations

Introduction: Hypertension (HTN) represents one of the greatest burdens in human health and is associated with increased morbidity and mortality. Despite the availability of several antihypertensive drugs, most patients have suboptimal blood pressure (BP) control, thus underscoring the need for new therapeutic drugs. The natriuretic peptide receptor A (NPRA) possesses potent vasodilating, natriuretic and diuretic actions via activation by ANP or BNP and generation of its second messenger, cGMP. Recently, we reported the discovery of a small molecule positive allosteric modulator (PAM) for NPRA, MCUF-651, which enhances the ANP/BNP/NPRA/cGMP pathway (PNAS,2021). To date, the in vivo actions of MCUF-651 remains unknown. Here we tested the hypothesis that MCUF-651 would have BP lowering and renal enhancing properties in experimental HTN. Methods: A single IV bolus of MCUF-651 (10mg/kg; n=4) or vehicle (V; n=5) was injected into anesthetized spontaneous hypertensive rats (SHRs). Plasma and urinary cGMP (PcGMP and UcGMP), mean arterial pressure (MAP) and urinary volume (UV) and sodium (UNa) excretion were assessed at baseline (BL) and over 60-mins post-bolus. Results: In SHRs, PcGMP and UcGMP were significantly elevated with MCUF-651 compared to the vehicle group (ΔPcGMP MCUF-651: 16±5, ΔPcGMP V: -2±3, pmol/mL; P =0.003; ΔUcGMP MCUF-651: 78±29, ΔUcGMP V: 30±8, pmol/min, P =0.01), thus supporting NPRA target engagement. Notably, the elevations in cGMP with MCUF-651 were associated with a significant reduction in MAP at 15-mins post bolus compared to vehicle (ΔMAP MCUF-651: -29±14, ΔMAP V: -6±2 mmHg; P =0.04) and this MAP reduction was sustained over 60 mins. Further, UV and UNa excretion were significantly increased, from BL, with MCUF-651 (BL UV: 6 ± 2, 60 mins post bolus UV: 56± 18 μL/min, P <0.001; BL UNa: 1±0.2, 60 mins post bolus UNa: 8±2 μmol/min, P <0.001). Conclusions: We report for the first time that MCUF-651, a novel NPRA small molecule PAM, engages NPRA, increases cGMP and reduces MAP while enhancing sodium and water excretion in experimental HTN. These findings support the development of MCUF-651 as a potential new therapeutic modality to optimize BP lowering goals and reduce adverse outcomes in human HTN.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII–NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Abstract Optimum function of HLA-DR molecules in transgenic mice requires efficient interaction between the class II molecules on APCs and CD4 on T cells. Residues 110 and 139 of the second domain of class II molecules are considered to be critical for recognition of CD4. We generated an HLA-DR4β(NT) transgene construct in which positions 110 and 139 were altered to resemble endogenous mouse H2 Aβ molecules. This construct was introduced into (B10 × SWR) embryos, and DR4β(NT) transgenic mice were produced. The transgene was transferred into B10.RFB3 (Eβ0 Eαp) mice. The transgene-encoded DR4β molecules paired with endogenous Eα chains to form stable DR4β/Eα dimers expressed on the cell surface. The hybrid dimers showed similar Ag-binding specificity to HLA-DR4 molecules and positively selected CD4+ T cells in vivo. Immunization of HLA-DR4β(NT) transgenic mice with DR4-restricted peptides induced T cell proliferation in vitro. While the purified T cells from DR4β(NT) transgenic mice responded strongly to the HA(307–319) presented by M12C3 transfectants expressing altered DR4β/Eα heterodimers, the response to the same peptides presented by transfectants expressing wild-type DR4β/Eα molecules was substantially reduced. Taken together, these data confirmed in vitro studies on the importance of these residues in CD4-MHC class II interaction. The altered HLA-DR4β transgenic mice were able to overcome the species barrier and generate efficient HLA-DR4-restricted CD4-specific immune responses. Thus, residues 110 and 139 were critical for the interaction of class II with CD4 T cells during thymic selection as well as peripheral immune responses.

Introduction: Therapeutic strategies dual-targeting renin-angiotensin system (RAS) and natriuretic peptide system (NPS), such as Sacubitril/Valsartan, have been proven promising in treating cardiorenal disease (CRD). However, mechanisms behind the crosstalk between RAS and NPS remain to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the receptor activated by ANP and BNP to increase its second messenger cGMP, has not been investigated. Hypothesis: We hypothesize ANGII naturally suppresses pGC-A activity and inhibition of this ANGII mediated action can be a new strategy to combat CRD. Methods: Studies consist of biomarker analyses in plasma from healthy humans, drug infusions in normal rats, molecular characterization in human HEK293 cells, and in vitro assessment of CRRL290, an innovative dual-functional peptide designed to activate pGC-A and inhibit protein kinase C (PKC). Results: In 128 healthy humans, plasma cGMP was positively associated with ANP or BNP in low (≤ 4.5 pg/mL) but not those with high (> 4.5 pg/mL) ANGII levels. Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII. Interestingly, suppression effect of ANGII on pGC-A was only recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. We also found PKC, a known downstream target of AT1R, was a key mediator as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Accordingly, we designed and synthesized CRRL290. Notably, CRRL290 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in HEK293 cells. Conclusions: We demonstrate that ANGII is a natural suppressor of pGC-A via AT1R and PKC signaling. We also report for the first time that CRRL290, a novel bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Our study provides new insights into the RAS-NPS interaction, and highlights a novel and mechanism-based therapeutic avenue for treating CRD.

Significance The particulate guanylyl cyclase A receptor (GC-A), via its endogenous cardiac-derived hormones atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP), plays a pivotal role in maintaining intravascular volume, arterial pressure, cardiovascular structure, function, and, more recently, metabolic homeostasis. As such, therapies to potentiate GC-A signaling via peptide augmentation have yielded promising results in clinical trials. A major breakthrough in GC-A therapeutics would be a small molecule that sensitizes the GC-A receptor to endogenous ANP or BNP, which to date does not exist. Here, we report the development of an orally bioavailable, small molecule positive allosteric modulator of GC-A that exhibits therapeutic activity in human hypertension and heart failure ex vivo.

BACKGROUND: BNP is a cardiac peptide with vasodilatory, natriuretic and diuretic properties. Recent studies have suggested that its vasodilatory hypotensive properties may limit the renal actions of BNP, especially in patients with borderline low blood pressure. We have recently identified an alternatively spliced transcript for BNP (ASBNP) that includes a unique and distinct longer carboxyl-terminus consisting of 34 amino acids. Based upon preliminary studies, we generated a truncated form (ASBNP2.1) that contains the first 16 amino acids of the C-terminal of ASBNP. METHODS: We determined the cardiorenal and humoral actions of intravenous infusion of ASBNP2.1 at 2 pmol/Kg/min, 10 pmol/Kg/min and 100 pmol/Kg /min in 10 dogs with rapid ventricular pacing induced overt CHF (240 bpm for 10 days). * p<0.05 RESULTS: IV infusion of ASBNP 2.1 increased aquaresis (from 0.19±0.04 to 0.32±0.07, 0.46±0.11 and 0.39±0.09 ml/min*) without a significant change in urinary sodium excretion. Importantly, ASBNP 2.1 enhanced glomerular filtration rate (GFR), from 31±4 to 47±8, 69±10 and 56±9 ml/min*. These renal actions were associated with increases in urinary BNP*, ANP* and cGMP* excretion. BNP 2.1 did not have any systemic vasodilatory action resulting in no change in mean arterial blood pressure or cardiac-filling pressures even at the highest dose. There was not change in serum sodium concentration. CONCLUSION: We report for the first time that this novel peptide based upon ASBNP has potent aquaretic and GFR enhancing actions without the vasodilatory hypotensive properties in an experimental model of overt CHF. The lack of vasodilatation but with renal actions also suggest that the C-terminus plays a key role in the vascular actions of this peptide offering new insights into vascular-renal structure function of BNP and related peptides. This renal specific peptide may have potential therapeutic benefit in states of renal dysfunction with volume overload to enhance GFR and water excretion without the detrimental side effect of hypotension.

Abstract TFPI is an endothelium associated anticoagulant protein that directly inhibits tissue factor (TF) pro-coagulant activity and prevents disease produced by intravascular thrombosis. TFPI is produced in three alternatively spliced isoforms in mice, α, β and γ, that differ in domain structure and mechanism for association with endothelium. TFPIα has three Kunitz-type serine protease inhibitory domains and a basic C-terminal region. It produces anticoagulant activity by direct inhibition of factor Xa (fXa) via the second Kunitz domain (K2) and, in a fXa dependent manner, inhibition of TF-fVIIa via the first Kunitz domain (K1). The third Kunitz domain (K3) and C-terminal region do not directly inhibit proteolysis. However, alignment of sequences from various vertebrate species demonstrates that K3 has maintained a high degree of sequence conservation for over 430 million years suggesting that K3 maintains a physiological function. TFPIα indirectly associates with endothelium by binding to a glycosylphosphatidyl inositol (GPI) anchored protein and binds non-specifically to glycosaminoglycans. TFPIβ has K1 and K2 but lacks K3 and has a distinct C-terminal region that encodes a GPI-anchor attachment sequence. TFPIγ has K1 and K2 but lacks K3 and has a distinct C-terminal region that results in production of a secreted protein. It is not known if these structurally diverse forms of TFPI with different mechanisms for cell surface association have variable efficacy in their ability to inhibit TF activity in vivo. We characterized TFPI isoform expression in mouse tissues in order to define specific physiological functions for alternative splicing of TFPI and how it may hinder diseases mediated by intravascular TF activity. Message for TFPIα is ~16-fold more abundant than message for TFPIβ or TFPIγ in mouse tissues. In situ hybridization studies of mouse heart and lung found that the isoforms are expressed in the same pattern, primarily in endothelial cells. No cell or vascular bed exclusively producing a single isoform was identified. Western blot analyses revealed the somewhat surprising finding that TFPIβ is the predominant isoform produced by adult mouse tissues. Thus, protein synthesis of TFPI isoforms in mice is regulated at least in part at the level of protein translation. Mouse plasma TFPI levels increase only 10–20% following heparin infusion. These data contrast with the 2- to 4-fold increase in plasma TFPI observed following heparin infusion in humans. The presence of only small amounts of heparin releasable TFPI in mouse plasma is consistent with TFPIβ as the major isoform because TFPIβ is directly GPI-anchored to endothelium and lacks the highly basic C-terminal region that allows TFPIα to associate non-specifically with endothelium glycosaminoglycans. To our knowledge, these are the first data demonstrating TFPIβ production in vivo. In contrast to the adult tissues, western blot analysis of mouse placenta revealed that it produces primarily TFPIα. This finding led us to examine mouse fetal tissue to determine if there is developmental regulation of the production of different TFPI isoforms. These studies demonstrated that TFPIα and TFPIβ are produced in approximately equal amounts in E14.5 embryos. The relative proportion of TFPIα in mouse tissues then decreases during development and very little is produced at 12 weeks of age. The evolutionary conservation of TFPIα and its production in mouse placenta and embryonic tissues suggests that unique features of TFPIα, such as K3, the basic C-terminal region and/or its binding to the endothelial surface through a GPI-anchored co-receptor, perform specific functions in the regulation of intravascular TF activity during vasculogenesis/angiogenesis that are not performed by TFPIβ or TFPIγ.

Based on the cardiac hormone atrial natriuretic peptide (ANP) and its seminal role in blood pressure (BP) homeostasis, we investigated the chronic BP lowering actions of a novel ANP analog currently entering clinical trials for hypertension. Previous reports demonstrate that this analog MANP activates the guanylyl cyclase A receptor (GC-A) and results in more potent biological actions compared with ANP; thus, it may represent a new therapeutic drug for hypertension. A major goal of this study was to establish that chronic subcutaneous delivery of MANP is feasible and hypotensive together with cGMP effects. We investigated the BP-lowering and cGMP-activating actions of acute and chronic subcutaneous delivery in normal and hypertensive rats. Furthermore, we explored vascular mechanisms of MANP in human aortic smooth muscle cells (HASMC) and ex vivo in isolated arteries. In normal rats with a single subcutaneous injection, MANP promoted robust dose-dependent BP-lowering actions and natriuresis, together with cGMP activation. Most importantly in hypertensive rats, once-a-day subcutaneous injection of MANP for 7 days induced cGMP elevation and long-term BP reduction compared with vehicle. Mechanistically, in HASMC, MANP activated cGMP and attenuated angiotensin II-mediated increases in intracellular Ca2+levels while directly vasorelaxing arterial rings. Our study demonstrates for the first time the effectiveness of subcutaneous administration of MANP for 7 days and provides innovative, vascular mechanisms of BP regulation supporting its continued development as a novel therapeutic for hypertension.

Abstract Abstract 4319 C-type natriuretic peptide (CNP) is known to exert pleiotropic effects on a variety of cell types through binding to its receptor, natriuretic peptide receptor type B (NPR-B) and activation of the cytoplasmic intracellular guanylyl cyclase domain. Although nitric oxide-mediated activation of cyclic guanosine monophosphate (cGMP) is known to induce megakaryocyte apoptosis and regulate platelet activation, the effects of CNP on these cells is yet to be defined. In this study for the first time we have addressed the potential role of CNP and NPR-B in megakaryocyte and platelet biology. We identified NPR-B on murine megakaryocytes by immunostaining demonstrating co-localization of CD41 and NPR-B. CNP induced cGMP production in megakaryocytes compared to a mutant form of CNP containing a single amino acid substitution (Lbab) (67.24 ± 10.65 fmol/well vs. 17.18 ± 2.089 fmol/well; P<0.05). Subsequently, CNP was shown to reduce megakaryocyte colony formation compared to Lbab and this effect was dose-dependent: Lbab 500nM 26.75 ± 5.618 CFU-M vs. 15.25 ± 2.750 CFU-M for CNP 500nM and Lbab 1000 nM 27.50 ± 2.102 CFU-M vs. 12.25 ± 1.493 CFU-M for CNP 1000nM; P<0.05). Furthermore, CNP also resulted in smaller-sized colonies, with altered cell morphology. To verify these findings, wild-type C57/BL6 mice were treated with daily injections of either saline, 1uM CNP or 1uM Lbab for one week, following which megakaryocytes were isolated and cultured. Colony formation was diminished in mice injected with CNP compared to mice receiving saline or Lbab peptide (125.3 ± 18.54 CFU-M for saline; 107.0 CFU-M ± 20.04 for Lbab and 20.00 ± 3.674 CFU-M for CNP; P<0.05). In addition platelet numbers were measured in the aforementioned mice and the CNP treated mice had significantly lower platelet counts compared to the mice treated with Lbab (832.0 ± 13.19 ×103/ul for CNP vs. 778.0 ± 12.00 ×103/ul for Lbab; P<0.05). Taken together, these data indicate that CNP may play a role in the development, maturation and/or survival of megakaryocytes. Using flow cytometry we identified that 47% of CD41+ murine platelets co-express NPR-B. In addition, this expression of NPR-B was confirmed by immunocytostaining and western blotting in both human and murine platelets. Interestingly, despite the expression of NPR-B, and unlike megakaryocytes, CNP (similar to the mutant peptide) did not increase cGMP production in platelets (1.652 ± 0.016 fmol/well for CNP and 1.606 ± 0.01550 fmol/well for Lbab). Further, although preliminary results did not indicate that CNP alters platelet secretion or aggregation, exposure to the peptide profoundly inhibited adhesion of platelets to collagen (53 ± 5 adhered platelets with Lbab, 32 ± 3 with CNP, relative reduction 42%; P<0.05). In summary, these results provide for the first time evidence that the NPR-B receptor is expressed on both megakaryocytes and platelets. Our studies suggest that CNP might have both cGMP dependent and independent functions in megakaryocytes and platelets and further studies will assist in delineating the mechanisms by which CNP exerts its actions on these cells. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor and prothrombinase during early stages of the procoagulant response. It is produced in alternatively spliced isoforms. In humans, TFPIα is within platelets and released upon activation and also is present in plasma, while TFPIβ is bound to the endothelial surface. Mice differ from humans in that they do not have TFPIα in plasma, making them an optimal model system for study of the function of platelet TFPIα. Studies of murine hematopoietic cell TFPI using fetal liver transplantation models have found that platelet TFPI dampens platelet accumulation in a growing thrombus following vascular injury and alters bleeding severity in mice with hemophilia. However, results of these studies are potentially confounded by altered TFPI expression on other hematopoietic cells, such as monocytes. To better understand the function of platelet TFPI, murine models of total TFPIα deletion (TFPIα KO), of platelet-specific TFPIα deletion (pTFPIα KO) and platelet-specific TFPIα over expression (pTFPIα high) were produced and characterized. Methods: A TFPIα deleted mouse was generated by inserting a stop codon within exon 10 of the TFPI gene effectively blocking translation of the highly conserved basic C-terminal region of TFPIα, but not altering TFPIβ. The PF4-Cre transgene was bred into the floxed-TFPI(K1) mouse to produce mice specifically lacking platelet TFPIα. A transgenic mouse over expressing platelet TFPIα under control of the GP1b promoter was produced. Mice were characterized for platelet and plasma TFPI antigen by ELISA, for activity by TF-FVIIa inhibition and whole blood platelet aggregation, and for bleeding by tail clip. Results: All 3 strains were viable and reproduced. Platelet TFPI antigen was decreased approximately 25-fold in the TFPIα KO and pTFPIα KO mice and elevated approximately 5-fold in the pTFPIα high mice with corresponding changes in TF-FVIIa inhibitory activity. Plasma TFPI antigen was no different between pTFPIα KO (20.6±4.4 nM) and wild type (21.5±6.2 nM) littermates. Plasma thrombin-antithrombin complex levels were not elevated in any strain. In tail clip bleeding time assays, there was no difference between pTFPIα KO (0.23±0.16 mg/ml Hb) and wild type (0.23±0.37 mg/ml Hb), but there was a trend towards increased blood loss in pTFPIα high mice (1.25±1.8 mg/ml Hb). The pTFPIα high mice also had altered whole blood platelet aggregation that was evident primarily in a prolonged time for initiation of aggregation with 6.50uM ADP (81±12 sec vs. 6.1±5.6 sec for control) or 0.8ug/ml collagen (35±17 sec vs. 16±21 sec for control). Finally, the pTFPIα high transgene was bred into TFPI heterozygous mice which were then bred to determine if over expression of platelet TFPIα would rescue the TFPI null embryonic lethal phenotype. However, no live TFPI null pups were observed following genotyping of over 50 pups from these matings. Conclusions: Three new mouse models of altered TFPI expression have been made providing valuable tools to study the role of platelet TFPI in bleeding and clotting disorders. Plasma TFPI is unchanged in mice lacking platelet TFPI demonstrating that platelet TFPI does not contribute to plasma TFPI in mice. Platelet TFPIα antigen and activity correlate in all three models. Deletion of the basic C-terminal region in TFPIα in the TFPIα KO mice produces a TFPIα hypomorphic mouse, with only trace amounts of truncated TFPIα present in platelets. This lack of TFPIα does not produce a generalized procoagulant state. The 5-fold excess TFPIα antigen in the pTFPIα high mice substantially delays the onset of platelet aggregation in whole blood assays initiated with collagen or ADP. Results from tail clip bleeding assays suggest that over expression of platelet TFPIα may prolong bleeding in adult mice, but it is not sufficient to rescue the embryonic lethality of TFPI null mice. Disclosures Mast: Novo Nordisk: Honoraria, Research Funding.